A motor neuron-like spheroid 3D model for in vitro evaluation of neurodegeneration

Three-dimensional (3D) spheroid models provide a bridge between traditional two-dimensional (2D) cultures and the complex in vivo tissue environment. These models are developed using self-clustering cells to mimic an appropriate 3D extracellular matrix (ECM). The NSC-34 cell line, created by fusing mouse spinal cord motor neurons and neuroblastoma cells, serves as a widely accepted model for studying motor neuron diseases. However, the precise behavior and features of this cell line in a 3D context have not been thoroughly investigated. In this work, we successfully generated NSC-34 spheroids using a non-adhesive agarose-based micro-wells template. Over five days, we examined the formation, stability, and morphological properties of spheroids by light and electron microscopy imaging and flow cytometry analysis. Spheroids with an initial density equal to 25000 cells showed a diameter size of 377±13 µm, a high circularity index (0.91±0.07) and a viability of ~85% after 5 days in culture. Our extensive ultrastructural analysis demonstrated that NSC-34 cells in 3D adopt and maintain a distinct round-like shape, enabling a network of short yet stable membrane connections between cells. No significant differences were detected in the ultrastructural morphology of subcellular organelles compared to 2D cultures. To showcase the potential of this system for pathological studies, we utilized the NSC-34 spheroid model to investigate the influence of 3D structure and cellular assembly on the expression of GFP-tagged full length TAR-DNA-binding protein 43 (TDP-43), a known marker of ALS pathology. NSC-34 cell line showed nuclear expression of GFP-TDP-43 in the spheroidal construct, with levels comparable to standard monolayer cultures. Our results offer valuable insights into the behavior of the NSC-34 cell line in 3D environments and emphasize the potential utility of spheroid models for exploring motor neuron diseases and associated pathologies.

Research funded by Ministero dell’Università e della Ricerca (MIUR) – Italy (PRIN2020 – n. 2020PBS5MJ)